The present invention is directed to immunoassay methods and apparatus, and more particularly concerns an immunobead-flow cytometry method, apparatus, assay, device, system, kit, and the like for detecting and quantifying platelets, antigens, antibodies and the like.
Microsphere based assays using flow cytometry have been reported by several investigators after Horan et al. reported the use of polystyrene microspheres to detect serum rheumatoid factor in 1979).
The merger of bead assays with flow cytometry has been demonstrated in several clinical applications, e.g. detection of antibodies to CMV and herpes simplex; detection of antibodies to different components of the human immunodeficiency virus (HIV); detection of antibodies to several antigens of Candida albicans; detection of human anti-mouse antibody (HAMA) in transplant patients receiving OKT3; detection of circulating immune complexes and HIV antibody in immune complexes; and detection of two different antibodies to CEA.
Although interest has focused on the detection of antibodies and antigens in fluids, the use of other ligand systems and biological probes has been explored, e.g. competitive binding of antibiotics to DNA coated beads and detection of viruses.
Although the principals and advantages of fluorescent microsphere immunoassays have been discussed in the literature, applications in clinical lab testing have been relatively few despite the economics of time and cost inherent in this technology.
Within the realm of flow cytometric applications and ever increasing interest in bead based technologies, it has been difficult, if not impossible to conceive the applications potentially available. One of these applications involves the entire spectrum of clinical laboratory testing.
Within the field of the Invention for Anti-platelet antibody bead positive control comes a multitude of other applications for the testing of immunologic material utilized in analytical testing. Any antigen, antibody, protein, chemical or ligand and be tested for its presence or viability by application to a bead based substrate and tested using immunological principles. For example, in the current Field of Invention we are testing for human immunoglobulins on the surface of patient platelets in disease states associated with immune or non-immune thrombocytopenia. Unfortunately, the secondary markers added to patient platelets can not be optimally tested for it's reactivity because of the lack of human platelets coated with a specific patient immunoglobulin for IgG, A, or M (e.g. a positive control). Therefore, the anti-Platelet positive control beads were designed to fill this void and to obtain a higher level of confidence that the anti-platelet antibody assay performed in the laboratory was utilizing reagents viable enough to justify the end result. To do this, latex beads were coated with human immunoglobulins, IgG, IgA, and IgM, thus mimicking the positive platelet itself. Within this system, bead size is dependent of the system you are testing them in. For platelet assays, the size of the coated bead must fall within 4 to 10 microns for easy detection without red cell, white cell or platelet size interference.
Applying this system to other assays with hard to find positive controls and/or antibody/antigen systems, any positive selection product can be made. Within this embodiment we demonstrate examples of positive control bead material for use with chemistry analytes, tumor markers, protein derivatives, and antibody/antigen systems. A confirmatory bead based substrate material has many applications in both clinical laboratories and industrial environments; especially when complex confirmatory material is hard to acquire. Furthermore, multiple bead sizes may be utilized, simultaneously, in the same reaction (assay) system to test multiple analyte integrity.